Tripterygium wilford II hook f extracts and components thereof for immunosuppression

ABSTRACT

The present invention involves the use of Tripterygium Wilfordii Hook F extracts in the treatment of rheumatoid arthritis. An alcohol extract of this plant (T2) inhibited antigen- and mitogen-stimulated proliferation of T cells and B cells, cell cycle progression, interleukin-2 (IL-2) production by T cells, immunoglobulin production by B cells and interleukin-2 mRNA production. T2 did not affect IL-2 receptor expression by T cells, IL-1 production by monocytes, the capacity of monocytes to present antigen, or signaling pathways. Inhibition could not be accounted for by nonspecific toxicity. These results support the conclusion that T2 exerts a powerful suppressive effect on human immune responses. Suppressing autoinnune disease is a most preferred embodiment of this invention.

The government has rights in the present invention as research relevantto the development thereof was supported by a grant from the UnitedStates government, NIH grant AR-36169.

This application is a continuation-in-part of application Ser. No.07/494,113 filed Mar. 14, 1990, now abandoned.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is a chronic inflammatory disease of uncertainetiology (1,2). Since the cause is unknown, treatment has been directedat suppressing the signs and symptoms of chronic inflammation. Althoughmany agents have been documented to decrease pain and swellingtemporarily, none has been shown to have a major impact on the course ofthe disease. New therapeutic modalities have been developed over thepast few years, employing monoclonal antibodies, cytokine antagonists,specific receptor targeted toxins, and other biologics (3-17).Nevertheless, uniform and persistent suppression of disease activity hasnot been reported. Although these approaches remain promising,alternative means of drug development seen warranted and could yield notonly new and effective treatment modalities, but also provide newinsights into disease pathogenesis that could serve as the basis offuture therapeutic innovations.

An area to search for new therapeutic interventions for RA is that oftraditional Chinese medicines. One of these traditional medicines isfrom Tripterygium wilfordii Hook F, a shrub-like vine from theCelastraceae family (18). A variety of preparations derived from thisplant have been used in South China for many years to treat differentforms of arthritis and other autoinmune diseases. In 1978, an extract ofTripterygium wilfordii Hook F was produced by chloroform reethanolextraction of the woody portion of the roots and designated T2 (18).Reports in the Chinese literature describe T2 treatment of more than 750patients with a variety of autoimmune diseases (19-35). The generalimpression has been that T2 is well-absorbed orally, appears to haveacceptable toxicity, and is effective in the treatment of variousautoimmune diseases.

T2 was evaluated in a double-blind placebo controlled cross-over studyinvolving 70 RA patients, with a mean disease duration of 6 years(19-20). Statistically significant improvement in all clinicalparameters, particularly ESR, CRP, and Rheumatoid factor titers, wasnoted after 12 weeks of therapy in the experimental group compared witheither baseline measurements or the placebo treated group. Of thepatients treated, 82-93% noted improvement in different clinicalcriteria or laboratory correlates of inflammation. An immunosuppressiveactivity was implicated by the finding that treatment induced inhibitionof the production of IgM and IgM rheumatoid factor by the patients'peripheral blood mononuclear cells in vitro (20). Toxicity, whichconsisted primarily of skin rash, gastrointestinal complaints andamenorrhea, was generally mild and reversible with cessation of therapy.These results support the contention that T2 might be an effectivetherapy for RA, but there is little experience with this agent outsideof China.

Tripterygium wilfordii Hook F is known to contain a number ofconstituents, some of which appear to be toxic (36). It is known thatthe leaves, stem, flowers, and the skin of the roots are poisonous andthat ingestion can cause death (37-39). In contrast, the woody portionof the roots of the plant is much less toxic. T2 is prepared from thewoody portion of the roots, appears to contain the therapeuticcomponents, and to have reduced toxicity compared with otherpreparations.

The Chinese experience has suggested that a daily dosage of 0.8-1.5mg/kg of T2 is safe and effective. Acute and chronic toxicity studieshave been carried out in China using a variety of animal models. TheLD₅₀ in mice is 159.7±14.3 mg/kg (40). The major chronic toxicity notedin rats administered 30 mg/kg for 90 days was azoospermia and decreasein testicular weight (40). Lower dosages of T2 did not cause decreasesin testicular weight. The toxicity studies, therefore, suggest that T2exhibits a reasonable safety index and should be able to be administeredto patients safely.

Research has begun in China to determine the spectrum of activity of T2.Triptonide and triptolide from this plant have been reported to inhibitthe proliferation of lymph cells induced by concanavalin A. ((Zhang etal., Shanghai Yike Da ue Xuebao, 1986, 13 (4) pp. 267-272.))Additionally, ancient Chinese medical books have suggested that thisherbal remedy is useful to treat joint pain. Recently, this extract hasbeen used in the treatment of rheumatic diseases including rheumatoidarthritis, as well as systemic lupus erythematosus, Behcet's disease,and psoriasis. Alcoholic extracts (T2) of the plant have been describedas having significant activity in vivo against certain mouse leukemiasand in vitro against cells derived from human carcinomas (Kupchan etal., J. An. Chem. Soc., 1972, 94 pp. 3194-3195). The capacity of T2 tosuppress a number of animal models of autoinmune disease, includingadjuvant arthritis and experimental allergic encephalomyelitis, has beenreported (41-47). Large concentrations of T2 (30 mg/kg) suppress delayedhypersensitivity reactivity in mice and may also suppress graft versushost disease, as well as skin and heart allograft rejection (36,41). Ingeneral, however, only very large concentrations of T2 have beenexamined in these studies. It, therefore, remains unclear whether lower,more pharmacologically appropriate concentrations would also exerttherapeutic effects in these animal models.

T2 is a crude extract containing a mixture of materials, includingvarious glycosides, alkaloids, and diterpenoids. The active principle,however, has not yet been identified. A few components have beenpurified, including triptolide, wilfordine, and related compounds, butproof that a particular purified component accounts for the therapeuticor immunosuppressive activity of T2 does not exist (48).

High concentrations of triptolide were reported to suppress B and Tlymphocyte proliferation and interleukin-2 production by mouse spleencells (Pu et al. (1990) Chem. Abstracts, 112:45, abstract 171972d). Theconcentrations used were sufficiently high that significant nonspecifictoxicity undoubtedly occurred.

SUMMARY OF THE INVENTION

The present invention involves the use of Tripterygium wilfordii Hook Fextracts (T2) or components thereof to selectively suppress the immunityof an animal or a patient in need of such treatment. Immunity of ananimal may include immunoglobulin synthesis, cell proliferation ofperipheral blood lymphocytes, cellular immune responses or proliferationof T and B lymphocytes.

In particular, the selective inhibition of interleukin-2 production byinhibition of IL-2 gene transcription and consequent inhibition of IL-2specific mRNA production without substantial cellular toxicity is anaspect of the present invention. Lack of substantial cellular toxicityis indicated by substantially unchanged interleukin-2 receptorexpression or cellular signaling activities such as inositoltriphosphate production, diacylglycerol generation, translocation ofprotein kinase C or protein tyrosine kinase activity. Lack ofsubstantial cellular toxicity by T2 may also ? Rean having little or noeffect on the capacity of monocytes to function as antigen presentingcells, having little or no effect on the growth of endothelial cells orfibroblasts, or having little or no effect on the viability of eitherresting or stimulated lymphocytes, endothelial cells, fibroblasts,monocytes or polymorphonuclear leukocytes.

The administration of Tripterygium wilfordii Hook F T2 extract in atherapeutically effective amount to suppress autoinmune disease in apatient in need of such treatment is a most preferred embodiment of thisinvention. A therapeutically effective amount of T2 inhibits IL-2production without substantial cellular toxicity. An in vivotherapeutically effective amount of T2 for humans is about 60 mg/day. Anin vitro therapeutically effective amount of T2 for cell cultures isabout 1.0 μg/ml. Particular autoimmune diseases thought amenable to suchtreatment include rheumatoid arthritis, systemic lupus erythematosus andpsoriasis.

A method of testing for selective inhibition of IL-2 specific mRNAproduction is an aspect of the present invention, the method consistingessentially of: culturing eukaryotic cells in culture with andseparately without Tripterygium wilfordii Hook F T2 extract orcomponents thereof in a therapeutically effective amount to provide atest sample and a control sample; measuring IL-2 aMA level and areference mRNA level such as actin mRNA to provide a test IL-2 mRNAsample, a test reference mRNA sample, a control IL-2 mRNA sample and acontrol reference mRNA sample; comparing (test IL-2 mRNA level÷controlIL-2 mRNA level) to (test reference mRNA level÷control reference mRNAlevel); wherein when (test IL-2 mRNA level÷control IL-2 mRNA level) issubstantially less than 1 and (test reference mRNA level÷controlreference mRNA level) is about 1, selective inhibition of IL-2 mRNAproduction by T2 is indicated.

The specific T2 impairment of IL-2 mRNA production with nontoxicity toother cellular functions and other cell types is a surprising andunexpected aspect of the present invention. This specificity of T2 canbe ascribed to individual or a combination of components of the T2extract.

    ______________________________________                                        ABBREVIATIONS                                                                 ______________________________________                                        CRP =    cross-reacting protein                                               DAG =    diacylglycerol                                                       ESR =    erythrocyte sedimentation rate                                       FACS =   fluorescence-activated cell sorter                                   Ig =     immunoglobulin                                                       IL-2 =   interleukin-2                                                        IL-2R =  interleukin-2 receptor                                               IP =     phosphatidyl inositol triphosphate                                   MAb =    monoclonal antibodies                                                NHS =    normal human serum                                                   PBMC =   peripheral blood mononuclear cells                                   PDB =    phorbol dibutyrate                                                   PHA =    phytohemagglutinin                                                   PKC =    protein kinase C                                                     RA =     rheumatoid arthritis                                                 SA =     formalinized Staphylococcus aureus                                   SK =     streptokinase                                                        SRBC =   sheep red blood cells                                                T2 =     an ethanol extract from the woody portion of                                  Tripterygium wilfordii Hook F                                        TT =     tetanus toxoid                                                       ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of T2 on T cell proliferation. T cells (1×10⁵ /well) werecultured with medium (□) or PHA (Δ) in the presence or absence ofvarying concentrations of T2 as indicated for 3 days. Results representthe mean cpm±SEM of three experiments.

FIGS. 2A-2L. Effect of T2 on cell cycle progression of human T cells. Tcells (1×10⁵ /well) were cultured with or without PHA (1 μg/ml) in theabsence or presence of the indicated concentrations of T2 for 24, 48 or72 hrs. The samples were harvested, stained with acridine orange, andanalyzed with an ORTHO flow cytometer using the CICERO prograra todetermine the position of cells in the cell cycle as assessed by theirRNA and DNA content.

FIGS. 3. Inhibitory effect of T2 on IL-2 production. T cells (1×10⁵/well) were cultured with medium (□) or PHA (Δ) in the presence orabsence of varying concentrations of T2 for 36 hrs. The cell-freesupernatants were diluted 1:4 and analyzed for IL-2 activity with CTLL-2cells. Mean [³ H]-TdR incorporation ±SEM of CTLL-2 cells from 6experiments is shown.

FIG. 4. Effect of supplemental IL-2 on T2 mediated inhibition of T cellproliferation. T cells (1×10⁵ /well) were stimulated with PHA with (□)or without ( ) IL-2 (10 U/ml) and in the presence or absence of varyingconcentrations of T2 for 3 days. The data are expressed as percent ofcontrol [³ H]-TdR incorporation from three experiments.

FIG. 5. Effect of T2 on steady state levels of IL-2 mRNA in mitogenstimulated T cells. T cells (1×10⁶ /ml) were cultured with and withoutPHA in the presence or absence of T2 (1 μg/ml). After a 4-hourincubation, total RNA was isolated and IL-2 and actin mRNA levelsdetermined by Si nuclease protection as described (54).

FIGS. 6A-6B. Effect of T2 on B cell DNA synthesis and Ig production. InFIG. 6A FIG. 6B , B cells (5×10⁴ /well) were stimulated with SA (o) orSA+IL-2 (o), and in the right panel with SA+IL-2 in the presence ofvarying concentrations of T2. [³ H]-TdR was determined after a 5-dayincubation (left panel). Supernatants were harvested after a seven-dayculture and assayed for IgM ( ) and IgG (o) and IgA (o) content (rightpanel). Results are the mean±SEN of 3 experiments.

FIGS. 7A-7B. Effect of T2 on total IP generation by activated T cells.Fresh T cells (A) or Jurkat cells (B) were labeled with [³H]-myo-inositol overnight in the absence or presence of the indicatedconcentrations of T2. Total IP was determined as described in Example 2.An aliquot of each cell population was also stimulated with PHA for 24hours and supernatants assayed for IL-2 content using CTLL-2 cells (o).Data are the mean of three replicate experiments.

FIGS. 8A-8C. Effect of T2 on the generation of IP fractions by PHAactivated T cells. Jurkat cells were labeled with [³ H]-myoinositolovernight in the presence or absence of various concentrations of T2.Following a 5 minute incubation with 10 Mm LiCl, the cells wereactivated with PHA for 60 min. Water soluble IPs were isolated (IP1,FIG. 8A ; IP2, FIG. 8B and IP3, FIG. 8C ) and quantitated as describedin Example 2. Data are from one of three similar experiments.

FIG. 9. Effect of T2 on DAG generation and IL-2 secretion by PHAstimulated T cells. DAG and IL-2 were assayed as described in Example 2.Data represent the mean of duplicate determination of three similarexperiments.

FIGS. 10A-10B. Effect of T2 on translocation of PKC. PKC activity inboth the cytoplasmic FIG. 10A and membrane FIG. 10B fractions wereassayed as described in Example 2.

FIG. 11. Effect of T2 on Protein tyrosine phosphorylation. See Example 2for methods. Arrows indicate tyrosine phosphorylation of new proteinsafter PHA stimulation.

FIG. 12 summarizes assessment of symptomatic improvement in rheumatoidarthritis patients as a result of treatment with a mixture fromTripterygium wilfordii Hook F.

FIG. 13 schematically shows the structure of triptolide (1) andtriptodiolide (2).

FIG. 14 schematically shows the structure of triptonide.

FIG. 15 schematically describes the structure of wilfortrine (1) andwilfortrine methyl ester (2).

FIG. 16 shows the structure of triptophenolide (1) and triptophenolidemethyl ester (2).

FIG. 17 schematically shows the structure of triptonoterpenol.

FIG. 18 schematically shows the structure of wilformine.

FIG. 19 outline the extraction procedure for preparation of triptolide.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention concerns the use of Tripterygium wilfordii Hook Fextracts to suppress immune function, particularly for the treatment ofautoimmune diseases. T2 was prepared by ethanol extraction ofTriptexygium wilfordii Hook F roots.

Example 1 concerns studies on the effect of T2 on human lymphocytefunction. Interleukin-2 production by T cells is inhibited by T2 due toan inhibition of gene transcription while the expression of IL-2receptors is not affected. T2 also suppresses proliferation of B cellsand immunoglobulin production by B cells. The experiments described inExample 2 indicate that signaling pathways are not affected by T2demonstrating the selective nature of T2 inhibition. Example 3 concernsresults of studies on the effect of T2 in the treatment of patients withrheumatoid arthritis.

EXAMPLE 1 Effect of T2 on human lymphocyte function

This example describes the effect of T2 on in vitro immuneresponsiveness of human peripheral blood mononuclear cells (PBMC)obtained from normal individuals. It was found that T2 exerted aconcentration-dependent profile of suppressive activity on both T celland B cell functions, whereas the functional activities of monocyteswere more resistant to the suppressive effects of T2.

Methods

Cell preparation. PMBC were obtained from the blood of healthy adults bycentrifugation on sodium diatrizoate/Ficoll gradients (Sigma, St. Louis,MO). Monocutes were isolated from PBMC by centrifugation onSepra-cell-MN (Sepratech, Oklahoma City, OK) or by glass adherence. Themonocytes obtained from the two procedures were used to examineinterleukin-1 (IL-1) production and antigen presentation, respectively.For purification of T cells and B cells, PBMC were incubated withLleucine methyl ester HCl (Sigma) for 45 minutes at room temperature todeplete monocytes and natural killer cells, (Thiele et al., J. Immunol.131:2282-2290, 1983). The resultant lymphocytes were rosetted withneuraminidase-treated sheep red blood cells (SRBC) and were thenseparated by Ficoll/diatrizoate centrifugation, (Rosenberg, et al., J.Immunol. 122:926-931, 1979). T cells were further purified by passage ofthe rosette-positive population over a nylon-wool column to removeresidual B cells and monocytes, (Rosenstreich, et al., J. Exp. Med.134:1170-1186, 1971). B cells were prepared from the initial populationof rosette-negative cells by removing any remaining cells that formedrosettes with neuraminidase-treated SRBC.

Staining with monoclonal antibodies (MAb) to CD3 and CD20 and analysiswith the fluorescence-activated cell sorter (FACS) indicated that the Tcell and B cell populations were more than 96% and 90% pure,respectively. T cells were incubated with mitomycin c (0.1 mg/ml) for 45minutes and then washed thoroughly, (Jelinek et al., J. Immunol.136:83-92, 1986).

Reagents, T2, an chloroform/extract from the woody portion of the rootsof TWH, was a kind gift of Taizhou Pharmaceutical Company (Taizhou,Jiang Su, People's Republic of China). It contained more than 8different compounds including glycosides, diterpenoids, alkaloids, andketones. Before use, the extract was dissolved in DMSO and furtherdiluted with culture medium. Phytohemagglutinin (PHA; Wellcome Reagents,Research Triangle Park, NC), phorbol dibutyrate (PDB; Sigma), ionomycin(Calbiochem, San Diego, CA), and the anti-CD3 MAb, 64.1, were used for Tcell activation (Geppert et al., J Immunol 138:1660-1666, 1987). MAb64.1 was purified as previously described [Hansen et al., "T cellprotocol", Leukocyte Typing. Edited by Bernard, et al. Berlin,Springer-Verlag, 1982]. Human recombinant interleukin-2 (rIL-2; Cetus,Emeryville, CA) and/or formalinized Staphylococcus aureus (SA;Calbiochem) was used for B cell activation. The MAb against the a chainof the IL-2 receptor (IL-2R), anti-Tac, was the gift of Dr. ThomasWaldmann (NIH, Bethesda, ND) and was used to analyze IL-2R expression.Interleukin-1 (Cistron Technology, Pine Brook, NJ) was purchased forstandardization of the IL-1 assay. Affinity-purified goat anti-humanIgA, IgG, and IgM and similar antibodies conjugated to horseradishperoxidase were purchased from Tago (Burlingame, CA). Streptokinase (SK)and tetanus toxoid (TT) were purchased from Hoechst-Roussel (Somerville,NJ) and MCDC Biologics (Jazaaica Plain, MA), respectively.

Cell culture and assay of lymphocyte DNA synthesis. T cells (1×10⁵/well) or B cells (5×10⁴ /well) alone or B cells with mitomycinc-treated T cells (1×10⁵ /well) were cultured in RPMI 1640 medium(Hazleton Biologics, Lenexa, KS) supplemented with 10% fetal calf serum,penicillin G (200 units/ml), gentamicin (10 μg/ml), and L-glutamine (0.3mg/ml) in 96-well microtiter plates in a total volume of 200 μl, with orwithout the stimuli indicated, and in the presence or absence of variousconcentrations of T2. The final concentration of DMSO in culture was0.02-0.002%. This concentration of DMSO had no effect on any of theresponses analyzed.

For both T and B cell activation, immobilized anti-CD3 (NAb 64.1)stimulation was used. This MAb was immobilized by incubating 50 μl (5μg/ml) in each well for at least 2 hours at room temperature. The excesssoluble antibody was removed before cell culture (Hansen, supra). Cellswere cultured for the indicated duration, and then pulsed with 1 μCi of³ H-thymidine, (³ H-TdR; New England Nuclear, Boston, MA) for the last12 and 18 hours for T cell and B cell cultures, respectively. ³ H-TdRuptake was measured in a liquid scintillation counter. All data areexpressed as the mean counts per minute of 3 replicate determinations(Davis et al., J Immunol 137:3758-3767, 1986).

IL-1 production assay. Monocutes (1×10⁵ /well) were suspended in RPMI1640 medium with 1% normal human serum (NHS) and cultured with orwithout lipopolysaccharide (10 μg/ml) in the presence or absence ofvarious concentrations of T2 for 24 hours. The culture supernatants werecollected, and serial dilutions were assayed for IL-1 using C3H/HeJmurine thymocytes as described elsewhere (Moreno et al., J Immunol136:3579-3587, 1986). Concentrations of T2 contained in the dilutions ofsupernatants had no effect on DNA synthesis by C3H/HeJ thymocytes.

IL-2 production assay. T cells (1×10⁵ /well) were incubated with orwithout PHA (1 μg/ml) or immobilized anti-CD3 in the presence or absenceof various concentrations of T2 for 24 hours. Cell-free supernatantswere harvested, serial dilutions were made, and IL-2 content was assayedwith CTLL-2 cells as described previously (Gillis et al., J Immunol120:2027-2032, 1978).

IL-2R expression. T cells were cultured with or without the indicatedstimuli in the presence or absence of various concentrations of T2 for36 hours. After washing, the cells were stained with saturatingconcentrations of anti-Tac or a mouse IgG control MAb, followed byfluorescein isothiocyanate-conjugated goat anti-mouse Ig antibody(Cappel, West Chester, PA). The samples were fixed with 1%paraformaldehyde and analyzed with a FACSTAR (Becton Dickinson, MountainView, CA) flow cytometer, using a single-histogram statistics program(Davis, supra).

Measurement of Ig synthesis. The amount of IgG, IgA, and IgM in theculture supernatants of B cells stimulated with SA plus rIL-2 in thepresence or absence of T2 for 7 days was determined using anisotype-specific enzyme-linked immunosorbent assay method. Quantitationof the Ig in the supernatants was then determined by comparison with astandard curve. The sensitivity of the assay is 15 ng/ml for IgA andIgG, and 30 ng/ml for IgN (Splawski et al., J Immunol 139:1432-1437,1986).

Results

Effect of T2 on human T cell responsiveness. These experiments notedthat T2 caused concentration dependent inhibition of PHA induced ³H-thymidine incorporation by purified human T lymphocytes (FIG. 1).Fifty percent inhibition was noted at concentrations of approximately0.2 μg per ml. Cell cycle analysis indicated that T2 prevented cellsfrom progressing through the Gl phase of the cell cycle (FIG. 2).Mitogen induced IL-2 production by purified T Cells was also inhibitedby a similar concentration of T2 (FIG. 3). Mitogen induced expression ofIL-2 receptors was not inhibited by T2 (Table I) indicating that T2 isnontoxic to this cellular activity. These results suggested that thedecrease in proliferation might be the result of inhibition of IL-2production.

                  TABLE 1                                                         ______________________________________                                        EFFECT OF T2 ON                                                               INTERLEUKIN-2 (IL-2) RECEPTOR EXPRESSION*                                     Nil                 PHA                                                               %        Fluorescence       Fluorescence                              T2      positive intensity  % positive                                                                            intensity                                 ______________________________________                                          0 μg/ml                                                                          10 ± 2                                                                              483 ± 18                                                                              65 ± 17                                                                            561 ± 166                              0.65 μg/ml                                                                         --       --         60 ± 22                                                                            519 ± 109                              1.25 μg/ml                                                                          9 ± 2                                                                              504 ± 29                                                                              61 ± 20                                                                            525 ± 128                              2.50 μg/ml                                                                         --       --         --      --                                        ______________________________________                                         *T cells (1 × 10.sup.5 /well) were cultured with medium or              phytohemagglutinin (PHA) in the presence or absence of various                concentrations of T2 as indicated for 36 hours. Cells were collected,         stained with antiTac monoclonal antibody followed by fluorescein              isothiocyanateconjugated goat antimouse IgG, and analyzed by flow             cytometry. Values are the mean ± SEM of 6 experiments.                

In order to examine IL-2 production, experiments were carried out inwhich the effect of T2 on proliferation was examined in the presence ofsupplemental IL-2. As can be seen in FIG. 4, much of the inhibitoryeffect of T2 was overcome by supplemental IL-2. These results suggestedthat one of the major actions of T2 was to inhibit IL-2 production. Thisappeared to result from an inhibition of IL-2 gene transcription sinceT2 inhibited the appearance of MMA for IL-2, as shown in FIG. 5. Theseexperiments confirmed that one action of T2 was to inhibit IL-2production.

Effect of T2 on human B lymphocyte responses.

Additional effects of T2 were demonstrated when its action on human Bcell responses was examined. As can be seen in FIGS. 6A-6B, T2 inhibitedboth mitogen-induced proliferation of highly purified B cells, as wellas immunoglobulin production in a concentration dependent manner. Theseresults suggested that T2 had additional effects beyond altering IL-2production. Some specificity for the action of T2 was demonstrated,however, when its effects on a number of other cell types were examined.Thus, T2 had no effect on IL-1 production by human monocytes nor ontheir capacity to function as antigen presenting cells (49). Inaddition, T2 had no effect on the growth of endothelial cells orfibroblasts during a 48 hour culture. None of the inhibitory effects ofT2 could be accounted for by non-specific toxicity, as inhibitoryconcentrations of T2 had no effect on the viability of either resting orstimulated lymphocytes, endothelial cells, fibroblasts, monocytes, orpolymorphonuclear leukocytes. These results support the contention thatT2 has a limited spectrum of immunosuppressive activity which cannot beaccounted for by non-specific toxic effects. Of importance, the capacityof T2 to suppress both IL-2 production by T cells, and proliferation andimmunoglobulin production by B cells could well explain the action ofthis agent in patients with RA.

EXAMPLE 2

Effect of T2 on critical signaling pathways.

The mechanism by which T2 might inhibit IL-2 production was examined ingreater detail. The possibility was explored that T2 might inhibit acritical signaling pathway involved in inducing transcription of theIL-2 gene. Current information suggests that T cell receptor occupancyleads to activation of tyrosine kinases, followed by stimulation ofphospholipase C. This results in production of phosphatidyl inositoltriphosphate and diacylglycerol, that induce increases in intracellularcalcium and activation of protein kinase C, respectively (50, 51, 52).Therefore, experiments were carried out to examine the possibility thatT2 might inhibit one of these signaling pathways.

Methods

Effect of T2 on total IP generation by activated T cells. Fresh T cells(A) or Jurkat cells (B) were labeled with [³ H]-myoinositol overnight inthe absence or presence of the indicated concentrations of T2. The cellswere washed and incubated with 10 mM LiCl for 5 minutes then activatedwith PHA for 60 min. The cells were extracted with 0.75 ml of a 1:1mixture of chloroform and reethanol, followed by 0.25 ml of chloroformand 0.25 ml of water. The phases were separated by centrifugation andthe water soluble fractions were applied to a 0.25 ml Agl-X8 formate ionexchange column. Total inositol phosphate was eluted with 1.5 ml of 0.1Mformic acid and 1M sodium formate. The radioactivity was quantified byscintillation counting. An aliquot of each cell population was alsostimulated with PHA for 24 hours and supernatants assayed for IL-2content using CTLL-2 cells.

Effect of T2 on the generation of IP fractions by PEA activated T cells.Jurkat cells were labeled with [³ H]-Myoinositol overnight in thepresence or absence of various concentrations of T2. Following a 5minute incubation with 10 mM LiCl, the cells were activated with PHA for60 min. Water soluble IPs were isolated and quantitated. To accomplishthis, the cultures were extracted with 0.75 ml of a 1:1 mixture ofchloroform/reethanol, followed by 0.25 al each of chloroform and water.The phases were separated by centrifugation and the water solublefraction was applied to a 0.25 ml Agl-X8 formate ion-exchange column,and washed extensively with 5 mM cold myoinositol. IP1, IP2 and IP3 weresequentially eluted with 4 ml of 0.2M ammonium formate plus 0.1M formicacid, 10 ml of 0.4M ammonium formate plus 0.1M formic acid and 10 ml of1M ammonium formate plus 0.1M formic acid respectively. Theradioactivity of the various elution fractions was quantified byscintillation counting.

Effect of T2 on DAG generation and IL-2 secretion by PEA stimulated Tcells. T cells for each sample were cultured overnight with PHA in thepresence or absence of the indicated concentrations of T2. The cellpellets were lysed with a mixture of chloroform and reethanol, andfractions separated with 1M NaCl and chloroform. The organic phase wascollected and dried under nitrogen. DAG mass in the organic extract wasassayed by solubilizing the lipid residues in a mixture of ³² p-γ-ATPand DAG kinase and phosphatidic acid, incubating at 37° C. for 1 hourduring which DAG was quantitatively converted to p-phosphatidic acid.The samples were dried and redissolved in chloroform. The solvent wasapplied to a silica gel and separated by thin layer chromatography withchloroform/methanol/acetic acid. After visualization with iodine, thespot which contained phosphatidic acid was harvested and radioactivitydetermined by liquid scintillation counting. An aliquot of cells wasalso stimulated with mitogen and supernatants harvested after 24 hoursand assayed for IL-2 content.

Effect of T2 on tranalocation of PKC. Jurkat cells (1×10⁶ /ml) wereincubated overnight with or without T2 at the indicated concentrations.The cells were lysed by sonication and then cytoplasmic and membranefractions separated by centrifugation. PKC activity in both thecytoplasmic and membrane fraction was assayed using a protein kinase Cassay system (Amersham) which employed a synthetic peptide as aphosphate acceptor in the presence of phosphatidylserine, calcium andPMA.

Effect of T2 on protein tyrosine phosphorylation. Jurkat cells (3×10⁶)were incubated overnight in the absence or presence of the indicatedconcentrations of T2. The cells were washed and stimulated with PHA for30 minutes. After centrifugation, the pelleted cells were solubilizedwith 1 x SDS sample buffer containing protease inhibitors. The lysateswere centrifuged at 10,000 rpm for 15 minutes. The supernatants wereanalyzed for protein phosphorylation by western blotting using a mousemonoclonal antibody (Upstate Biotechnology, Inc.) againstphosphotyrosine.

Results

The effect of T2 on mitogen induced production of phosphatidyl inositolmetabolites. As can be seen in FIG. 7, mitogenic stimulation lead to theproduction of IL-2 and phosphatidyl inositol metabolites. Whereas IL-2production was inhibited, generation of phosphatidyl inositolmetabolites was not. Similar results were seen in fresh T cells and inthe Jurkat leukemic T cell line. Additional experiments examined whetherT2 specifically inhibited generation of IP3, which is thought to induceincreases in intracellular calcium (52). As can be seen in FIG. 8, T2had no effect on the generation of IP3 or other specific PI metabolitesby mitogen activated T cells. Similar experiments examined the effect ofT2 on the generation of diacylglycerol. As can be seen in FIG. 9, T2inhibited IL-2 production from mitogen stimulated T cells, but had noeffect on DAG production. Additional experiments, not shown, examinedthe activity of T2 on phospholipase C activity isolated from fresh Tcells or Jurkat cells. Again, no inhibitory activity was observed. Theseexperiments suggested that the action of T 2 cannot be explained by aneffect on these early signaling pathways. At these levels of T2 extractaddition, nontoxicity to other cellular functions is established asindicated by these cellular assays.

The effect of T2 on protein kinase C activation. As can be seen in FIG.10, mitogen stimulation led to translocation of PKC in Jurkat cells, andT2 did not effect PKC translocation. Finally, the effect of T2 on theactivity of protein tyrosine kinase activity was explored. As can beseen in FIG. 11, mitogenic stimulation of T cells lead tophosphorylation of a number of protein species identified with aspecific antibody to phosphotyrosine. However, T2 did not inhibit theactivity of protein tyrosine kinase since the same bands were observedregardless of the presence of T2 during mitogenic stimulation. Theseexperiments convincingly demonstrate that T2 has no effect on earlysignaling pathways involved in induction of IL-2 gene transcription.

EXAMPLE 3

In a preliminary open trial, it was found that a mixture of compounds(T2) extracted from Tripterygium wilfordii Hook F was effective in thetreatment of rheumatoid arthritis.

To confirm the previous results obtained from these open studies, aprospective, controlled, double-blind cross-over study was designed andcarried out in the outpatient clinic of Dr. Tao in Beijing, People'sRepublic of China.

The treatment plan was designed as follows:

Seventy patients with classic or definite adult-onset rheumatoidarthritis who had active disease for more than 6 months were acceptedinto the trial and randomly assigned to 2 treatment groups. Patients inGroup A received T2 for a first course of treatment of 12 weeks, andthen were subsequently changed to placebo for a second course oftreatment of 4 weeks duration. Patients of Group B received placeboduring the first course and then were crossed-over and received T2therapy during the second course. T2 was taken in a dosage of 60 mgdaily. Placebo tablets were identical in appearance to T2 tablets. Table2 shows the treatment plan schedule.

                  TABLE 2                                                         ______________________________________                                        TREATMENT PLAN (TOTAL COURSE: 16 WEEKS)                                                First course treat-                                                                       Second course treat-                                              ment (12 weeks)                                                                           ment (4 weeks)                                           ______________________________________                                        Group A    T2, 20 mg t.i.d.                                                                            Placebo                                              Group B    Placebo       T2, 20 mg t.i.d.                                     ______________________________________                                    

All patients were assessed in an arthritis clinic every 4 weeks. Theclinical assessment, overall assessment by physicians and drugdistribution were carried out by individual doctors in a blinded manner.The laboratory assessments were done by technicians of a centralhospital laboratory, who were also blinded to the details of the trial.

                  TABLE 3                                                         ______________________________________                                        CLINICAL FEATURES                                                             OF PATIENTS ENTERING THE TRIAL                                                           First       Second                                                            Treatment Course                                                                          Treatment Course                                                  Group A                                                                              Group B  Group A  Group B                                              T2     Placebo  Placebo  T2                                        ______________________________________                                        Number of Patients                                                                         35       35       27     31                                      Male/Female  3/32     4/31     1/26   4/27                                    Mean age, years                                                                            46.3     48.0     46.2   47.7                                    Mean disease duration                                                                      5.9      6.1      5.8    6.0                                     (years)                                                                       Stage of Disease                                                              (1)          6        6        4      5                                       (2)          14       16       11     13                                      (3)          12       10       10     9                                       (4)          3        3        2      4                                       ______________________________________                                    

The clinical features of patients entering the trial are shown in Table3. Statistical analyses demonstrated that at the beginning of the trial,Group A and Group B did not differ from each other significantly in age,sex, duration of disease or stage of disease.

                  TABLE 4                                                         ______________________________________                                        RESULTS OF A CONTROLLED TRIAL OF                                              T2 IN RHEUMATOID ARTHRITIS                                                                      No. of                                                                Number  Patients Completing Treatment                                           Beginning First Course                                                                             Second Course                                Group       Treatment (12 wks)   (4 wks)                                      ______________________________________                                        A (T2 → Placebo)                                                                   35        27         24                                           B (Placebo → T2)                                                                   35        31         25                                           ______________________________________                                    

As shown in Table 4, 27 patients of Group A completed the first courseof treatment, of which 24 completed the second course. 31 and 25 ofGroup B completed the first course and second course of treatment,respectively.

Table 5 indicates the reasons patients withdrew from the study. Threepatients of Group B but none of Group A withdrew from the trial becauseof worsening of disease during the first course of treatment, whereas 4patients from Group A but none from Group B withdrew from the trialbecause of side effects.

                  TABLE 5                                                         ______________________________________                                        REASONS FOR WITHDRAWAL FROM THE STUDY                                                  First       Second                                                            Course Treatment                                                                          Course Treatment                                                  Group A                                                                              Group B  Group A   Group B                                             T2     Placebo  Placebo   T2                                                  (n = 35)                                                                             (n = 35) (n = 27)  (n = 31)                                            No.  %     No.    %   No.  %    No.  %                               ______________________________________                                        Lost to follow up                                                                        4      11    1    3   3    11   6    19                            Worsening of                                                                             0       0    3    9   0    0    0    0                             disease                                                                       Side effects                                                                             4      11    0    0   0    0    0    0                             ______________________________________                                    

Table 6 shows the therapeutic effects of the first course of treatment.In comparison with patients of Group B, patients of Group A showedsignificant improvement in all clinical assessments including morningstiffness, joint tenderness score, number of swollen joints, gripstrength and 15 meter walking time.

                  TABLE 6                                                         ______________________________________                                        CHANGES IN CLINICAL PARAMETERS IN PATIENTS                                    COMPLETING THE FIRST COURSE OF TREATMENT                                                     Group A Group B                                                               T2      Placebo                                                               (n = 27)                                                                              (n = 31)  *p                                           ______________________________________                                        Morning stiffness                                                                          Before   2.4 ± 0.4                                                                            1.1 ± 0.2                                                                         0.01                                   (hours)      After    0.9 ± 0.2                                                                            2.3 ± 1.4                                  Joint tenderness                                                                           Before  25.1 ± 1.9                                                                           25.5 ± 1.7                                                                          0.001                                 score        After    7.9 ± 1.3                                                                           21.9 ± 2.1                                  Number of swollen                                                                          Before   9.2 ± 0.9                                                                            7.8 ± 0.7                                                                         0.01                                   joints       After    4.3 ± 0.6                                                                            7.4 ± 1.1                                  Grip strength (mean                                                                        Before  49.0 ± 0.4                                                                           73.6 ± 7.7                                                                         0.05                                   of both sides, mm Hg)                                                                      After   84.4 ± 7.5                                                                           81.2 ± 8.9                                  15 meter walking time                                                                      Before  36.6 ± 6.6                                                                           37.0 ± 2.4                                                                         0.05                                   (second)     After   21.6 ± 1.5                                                                           31.9 ± 3.6                                  ______________________________________                                    

The most noteworthy improvement was observed in joint tenderness score,which improved from a mean of 25.1 before entry to a mean of 7.9 afterthe first course of treatment with T2. By contrast, there were nosignificant changes in this score in Group B patients treated withplacebo.

As shown in Table 7, treatment with T2 also caused improvement inlaboratory correlates of disease activity. Significant improvements inESR, CRP and immunoglobulin levels were noted. The changes weresignificant at the p 0.001 level when compared between Group A and GroupB.

                  TABLE 7                                                         ______________________________________                                        CHANGES IN LABORATORY PARAMETERS                                              IN PATIENTS COMPLETING                                                        THE FIRST COURSE OF TREATMENT                                                              Group A  Group B                                                              T2       Placebo                                                              (n = 27) (n = 31)   *p                                           ______________________________________                                        ESR (mm/hour)                                                                            Before  69.2 ± 6.4                                                                            63.9 ± 5.2                                                                          <0.001                                            After   41.0 ± 5.9                                                                            67.2 ± 6.6                                   CRP (u/ml) Before  29.4 ± 5.7                                                                            31.6 ± 4.1                                                                          <0.001                                            After   10.4 ± 3.9                                                                            43.7 ± 7.0                                   RF (titers)                                                                              Before   87.1 ± 23.2                                                                           86.1 ± 35.5                                                                        NS                                                After    48.0 ± 13.4                                                                           63.4 ± 10.9                                 IgG (u/ml) Before  227.5 ± 4.6                                                                           231.9 ± 14.2                                                                        <0.001                                            After   117.4 ± 9.5                                                                           180.4 ± 29.8                                 IgM (u/ml) Before  302.8 ± 40.3                                                                          284.5 ± 32.2                                                                        <0.001                                            After   105.2 ± 11.1                                                                          261.3 ± 29.3                                 IgA (u/ml) Before  289.6 ± 29.4                                                                          257.6 ± 25.2                                                                        <0.001                                            After   149.0 ± 15.5                                                                          280.4 ± 29.8                                 ______________________________________                                         *Group A vs Group B                                                      

There was a greater tendency to decrease RF titer in T2 treated patientsbut the difference between the two groups after the first course oftreatment was not statistically significant.

During the second course of therapy, patients who had received placeboinitially improved significantly after 4 weeks of therapy with T2. (SeeTable 8). Significant improvements in joint tenderness score, number ofswollen joints and grip strength were noted. Improvement in morningstiffness and 15 meter walking time were also noted, but these changesdid not achieve statistical significance. Patients who had received T2during the first 12 weeks of therapy continued to maintain improvementeven after 4 weeks of placebo therapy during the second course.

                                      TABLE 8                                     __________________________________________________________________________    CHANGES IN CLINICAL PARAMETERS IN PATIENTS COMPLETING                         THE SECOND COURSE OF TREATMENT                                                                Group A   Group B                                                             Placebo   T2                                                                  (n = 24)                                                                            *p  (n = 25)                                                                              *p                                          __________________________________________________________________________    Morning stiffness                                                                        Before                                                                              1.8 ± 0.2                                                                       NS   2.5 ± 1.7                                                                         NS                                          (hours)    After                                                                               0.8 ± 0.2                                                                            1.3 ± 0.9                                       Joint tenderness                                                                         Before                                                                              7.9 ± 1.4                                                                       NS  22.2 ± 2.4                                                                          <0.001                                     score      After                                                                              11.0 ± 2.6                                                                           13.5 ± 2.0                                       Number of swollen                                                                        Before                                                                              4.2 ± 0.8                                                                       NS   7.0 ± 1.2                                                                         <0.05                                       joints     After                                                                               4.4 ± 0.9                                                                            3.5 ± 0.5                                       Grip strength (mean                                                                      Before                                                                             87.5 ± 8.0                                                                       <0.05                                                                             80.1 ± 9.2                                                                         <0.05                                       of both sides, mm Hg)                                                                    After                                                                              70.2 ± 9.5                                                                            97.1 ± 13.2                                     15 meter walking time                                                                    Before                                                                             20.3 ± 1.7                                                                       NS  31.5 ± 5.9                                                                         NS                                          (seconds)  After                                                                              17.1 ± 0.6                                                                           18.9 ± 2.3                                       __________________________________________________________________________     *After vs before treatment                                               

Aside from grip strength, no significant changes were observed inclinical assessments in Group A patients after 4 weeks of placebotreatment.

As shown in Table 9, significant decreases in ESR and RF titer werenoted in Group B patients after the second course of treatment. Nosignificant worsening in laboratory parameters were noted in Group Apatients after 4 weeks of placebo therapy.

                  TABLE 9                                                         ______________________________________                                        CHANGES IN LABORATORY                                                         PARAMETERS IN PATIENTS COMPLETING                                             THE SECOND COURSE OF TREATMENT                                                           Group A        Group B                                                        Placebo        T2                                                             (n = 24)                                                                              *p     (n = 25)   *p                                       ______________________________________                                        ESR     Before   42.3 ± 6.0                                                                           NS   68.5 ± 6.9                                                                          <0.001                               (mm/hour)                                                                             After    31.7 ± 7.3  22.0 ± 4.9                                 RF (titer)                                                                            Before   49.3 ± 13.5                                                                          NS   67.2 ± 12.1                                                                         <0.05                                        After    32.0 ± 12.3 32.0 ± 19.1                                ______________________________________                                         *After vs before treatment                                               

The overall effectiveness of T2 in the present trial was classified byits capacity to induce remissions, meaningful improvement or notherapeutic effect. (See Table 10).

                  TABLE 10                                                        ______________________________________                                        OVERALL EVALUATION OF THE PRESENT TRIAL                                                              Second                                                          First Course Treatment                                                                      Course Treatment                                       Clinical Response                                                                        Group A   Group B   Group A                                                                              Group B                                 Compared to                                                                              T2        Placebo   Placebo                                                                              T2                                      The Beginning                                                                            (n = 27)  (n = 31)  (n = 24)                                                                             (n = 25)                                of the Trial                                                                             No.    %      No.  %    No.  %   No.  %                            ______________________________________                                        Remission   2     7.4    0     0    0    0   0    0                           Improvement                                                                   Patient's  25     93     7    23   20   82  20   80                           assessment                                                                    Physician's                                                                              25     93     7    23   19   79  22   88                           assessment                                                                    Clinical   22     82     7    23   19   79  11   44                           criteria                                                                      Laboratory 23     85     4    13   18   75  13   52                           evaluation                                                                    ______________________________________                                    

Based on the therapeutic criteria for remission in RA developed by asubcommittee of the ARA, remission was observed in two patients of GroupA at the end of the first course of treatment.

The percentage of patients who experienced meaningful improvements wassignificantly higher for Group A than for Group B patients as evaluatedby physician's assessment, and clinical and laboratory evaluations afterthe first course of treatment.

The percent of Group B patients experiencing meaningful improvementafter the second course of treatment was also remarkable, whereasimprovement was maintained in Group A patients during the 4 week secondcourse of placebo.

In order to determine whether T2 exerted an immunosuppressive effect inpatients with RA, peripheral blood mononuclear cells (PBMC) wereobtained from 18 patients of each group before and after the firstcourse of treatment. These cells were cultured for 14 days and theamounts of IgM-RF and total IgM secreted were determined using aradioimmunoassay. (See Table 11).

                  TABLE 11                                                        ______________________________________                                        PRODUCTION OF IgM-RF AND TOTAL IgM BY PBMC OF                                 PATIENTS AFTER THE FIRST COURSE OF TREATMENT                                          Group A     Group B                                                           T2          Placebo                                                           (n = 18)    (n = 18)   *p                                             ______________________________________                                        RF    Before   7.2 ± 3.2 5.4 ± 1.6                                                                           <0.01                                          After    1.5 ± 0.5 7.0 ± 2.2                                      IgM   Before  220.7 ± 53.6                                                                             260.5 ± 49.3                                                                        <0.01                                          After   151.9 ± 55.3                                                                             301.2 ± 100.5                                  ______________________________________                                         *Group A vs Group B                                                      

In comparison with Group B, significant decreases in both IgM-RF andtotal IgM were noted in Group A after T2 treatment. These resultssuggest that T2 therapy had suppressed both IgM and IgM RF production inthese patients and thus exerted an immunosuppressive effect.

As shown in Table 12, the most common side effects of T2 were dermalreactions including skin rash, cheilosis, thinning of skin and nails andpigmentation.

                  TABLE 12                                                        ______________________________________                                        INCIDENCE OF ADVERSE REACTIONS                                                         First       Second                                                            Course Treatment                                                                          Course Treatment                                                  Group A                                                                              Group B  Group A   Group B                                             T2     Placebo  Placebo   T2                                                  (n = 31)                                                                             (n = 31) (n = 24)  (n = 25)                                            No.  %     No.    %   No.  %    No.  %                               ______________________________________                                        Skin rash &                                                                              15     39    1    3   0    0    7    28                            cheilosis                                                                     Diarrhea   6      27    0    0   0    0    2    8                             Anorexia   2       5    0    0   1    4    0    0                             Abdominal pain                                                                           2       5    1    3   0    0    0    0                             Amenorrhea 5/16   31    0    0   5/16 31   1/18 6                             Postmenopausal                                                                           1/10   10    0    0   0    0    0    0                             vaginal bleeding                                                              ______________________________________                                    

Although the incidence of skin reactions was quite high in Group Aduring the first course of treatment, none of the patients had todiscontinue T2 treatment. Amenorrhea was another important side effectof T2. It was observed that 31% of female patients aged 49 or lesshaving received T2 for 12 weeks developed amenorrhea whereas 6% ofpatients developed it after 4 weeks of T2 treatment. Amenorrheadisappeared in most patients when T2 was discontinued.

FIG. 12 summarizes the assessed improvements in symptoms of rheumatoidarthritis described above. T2 is an effective treatment for rheumatoidarthritis, significantly improving clinical manifestations andlaboratory correlates of inflammation. Although toxicity was frequent,it necessitated cessation of therapy in few. clinical improvement wasobserved after only 4 weeks of therapy and persisted for at least 4weeks after the medication was discontinued. T2 therapy suppresses thein vitro production of IgM and IgM rheumatoid factor.

T2 administration has also been shown to be effective in the treatmentof systemic lupus erythematosus (Table 13). T2 appears to be effectivein relieving acute clinical manifestations including joint inflammation,skin rash and renal disease (Table 13). A steroid sparing effect of T2was also noted. In comparison with corticosteroids and commonly usedimmunosuppressive agents, such as cyclophosphamide, patients treatedwith T2 had fewer significant complications.

                  TABLE 13                                                        ______________________________________                                        THERAPEUTIC EFFECT OF T2 IN LUPUS NEPHRITIS                                   ______________________________________                                        1.   Patient group                                                                 10 patients, aged 22-37, with duration of disease >1 year                     were treated with T2                                                     2.   Laboratory evaluation - before treatment                                      +ANA: 10                                                                      anti-DNA binding >20%: 9                                                      Proteinuria >3 g/24 h: 10                                                     Elevated serum creatinine: 3                                             3.   Treatment plan:                                                               First month: T2 20 mg tid. Maintain prednisone <40                            mg/day                                                                        Followed by T2 10 mg tid. and tapered prednisone                              Total course of T2: 24 weeks                                             4.   Results of treatment:                                                         Serum creatinine returned to normal in 2/3                                    Proteinuria improved in 10/10:                                                undetectable: 3                                                               <1 g/24 h: 3                                                                  >1 g/24 h: 4                                                                  Concomitant Medication                                                        3: withdrew from prednisone                                                   6: continued prednisone <10 mg/day                                            1: changed to cyclophosphamide                                           ______________________________________                                    

The following references and those cited in the text are incorporated inpertinent part by reference herein for the reasons cited in thespecification.

EXAMPLE 4 Components of T2 extract and toxicity thereof

The structure of triptolide and triptodiolide are shown in FIG. 13. FIG.14 shows the structure of triptonide. Triptolide was isolated fromalcoholic extracts of Tripterygium wilfordii Hook F by the method ofKupchan et al. (J. Am. Chem. Soc. 94, 7194-7195, 1972). This scheme fortriptolide preparation is outlined in FIG. 19.

The effect of triptolide on immunopotent cells in vitro was determinedas follows:

T cells, B cells and fibroblasts (1×10⁶ /ml) were incubated with varyingconcentrations of T2 or triptolide for 72 hr. The cells were assayed forcell viability by using a cytoflowmeter (FACSCAN) after the cells werestained with propidium iodine. Table 14 demonstrates the effect of T2 ortriptolide on cell viability.

                  TABLE 14                                                        ______________________________________                                        Effect of T2 or Triptolide on Cell Viability                                  Inhibitors                                                                                            Triptolide                                                      T2 (μg/ml) (ng/ml)                                               Control     0.1    1.0    10.0 100.0                                                                              0.1  1.0  10.0                            Cell type                                                                            (Percent viable cells)                                                 ______________________________________                                        T cells                                                                              91.7     90.0   89.3 88.2 18.8 29.5 29.8 11.5                          B cells                                                                              55.6     50.9   44.3 30.5 10.6 20.9 20.9 15.6                          Fibro- 77.5     92.7   95.1 86.6 43.0 91.7 89.3 35.8                          blasts                                                                        ______________________________________                                    

T2 at 100 μg/ml and triptolide at 10 ng/ml were toxic to fibroblastsindicating that at these levels, toxicity is nonspecific. At lowerlevels, suppression of T cell and B cell function is seen.

The capacity of triptolide to inhibit in vitro responses of humanlymphocytes was examined. As can be seen in table 15, triptolideinhibited proliferation of both T and B lymphocytes profoundly atconcentrations of 0.1-1.0 ng/ml.

                  TABLE 15                                                        ______________________________________                                                   PHA-Induced T Cell                                                                            SA-Induced B Cell                                  Concentration of                                                                         DNA Synthesis   DNA Synthesis                                      triptolide (ng/ml)                                                                       (.sup.3 H-Thymidine Incorporation, CPM)                            ______________________________________                                        0          93,400          7,900                                              0.1        24,200          2,000                                              1.0          100             100                                              ______________________________________                                    

Additional experiments indicated that this triptolide fraction alsoinhibited the in vitro production of immunoglobulin from mitogenstimulated human B lymphocytes at comparably small concentrations. Theseresults support a conclusion that this triptolide fraction is extremelytoxic, however, its specificity of action is yet to be determined. othercomponents of Tripterygium wilfordii Hook F possibly having specificbiological activity and thought to be useful individually or incombination in the practice of the present invention include:

polpunonic acid (wilfortrine) (1) and the methyl ester thereof (2) shownin FIG. 15 and described by Keng et al. (Chen. Abst. 107:55718y, p436,1987);

triptophenolide (1) and triptophenolide methyl ether (2) shown in FIG.16 and described by Wu et al. (Chem. Abst. 107:96917f, p712, 1987);

triptonoterpenol shown in FIG. 17 and described by Deng et al. (Chem.Abst. 107:112684k, p112692, 1987); and

wilformine (shown in FIG. 18), wilforine, wilforgine, and wilforzinedescribed by He et al. (Chem. Abst. 107:130906p, 1987;

Purified cozaponents of T2 will be administered to patients withautoimmune and inflammatory diseases including rheumatoid arthritis,systemic lupus erythematosus and psoriasis. Dosage will be determinedbased on the concentration of each component in the crude T2 mixture.After phase I dosage; escalation studies are carried out to evaluatetoxicity, trials with non-toxic doses will be carried out to determineefficacy.

The following references are incorporated in pertinent part by referenceherein for the reasons cited in the specification.

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What is claimed is:
 1. A method for inhibiting interleukin-2-mediatedimmunosuppression comprising administering a preparation consistingessentially of a Tripterygium Wilfordii Hook F root extract in atherapeutically effective amount to a patient in need of such treatment;and inhibiting interleukin-2 mediated immunosuppression in the patient.2. A method for suppressing interleukin-2 in autoimmune diseasecomprising administering a preparation consisting essentially ofTripterygium Wilfordii Hook F root extract, in a therapeuticallyeffective amount to a patient having an autoimmune disease sufficient tosuppress interleukin-2 production.
 3. The method of claim 2 wherein theautoimmune disease is rheumatoid arthritis, systemic lupus erythematosusor psoriasis.
 4. A method of inhibiting interleukin-2 mediatedimmunoglobulin systhesis comprising administering a preparationconsisting essentially of a Tripterygium Wilfordii Hook F root extractin a therapeutically effective amount sufficient to inhibitinterleukin-2 production to a patient in need of such treatment.
 5. Amethod of inhibiting interleukin-2 mediated cell proliferation of humanperipheral blood lymphocytes comprising administering a preparationconsisting essentially of a Tripterygium Wilfordii Hook F root extractin a therapeutically effective amount sufficient to inhibitinterleukin-2 production to a patient in need of such treatment.
 6. Amethod of inhibiting interleukin 2-mediated cellular immune responsescomprising administering a preparation consisting essentially of aTripterygium Wilfordii Hook F root extract in a therapeuticallyeffective amount sufficient to inhibit interleukin-2 production to apatient in need of such treatment.
 7. A method of selectively inhibitingthe production of interleukin-2 comprising administering a preparationconsisting essentially of a Tripterygium Wilfordii Hook F root extractin a therapeutically effective amount sufficient to inhibitinterleukin-2 production to a patient in need of such treatment.
 8. Themethod of claim 1, 2, 4, 5, 6, or 7 wherein the therapeuticallyeffective amount is about 60 mg/day.
 9. The method of claim 1, 2, 4, 5,6 or 7 wherein the lack of substantial cellular toxicity is measured bysubstantially unchanged interleukin-2 receptor expression or cellularsignaling activity.
 10. The method of claim 9 wherein the cellularsignaling activity is inositol triphosphate production, diacylglycerolgeneration, translocation of protein kinase C or protein tyrosine kinaseactivity.